A protein nucleic acid nanoparticle and a method of preparing the same

Abstract

The application discloses a protein nucleic acid nanoparticle and a preparation method thereof. The application belongs to the field of biological medicine and specifically relates to a protein nucleic acid nanoparticle and a preparation method thereof. In the nanoparticle, the protein only needs to add a certain number of positive amino acids in the sequence, and through the method of reducing the salt concentration in the buffer, the deoxyribonucleotide or ribonucleotide with different lengths, single strands or double strands and different sequences can be assembled to form the nanoparticle with a size of about 30 nm. The three-dimensional structure of the amino terminal of the protein in the nanoparticle can be completely recognized, so that the corresponding function is exerted. Meanwhile, the nanoparticle can be taken up by cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a protein and nucleic acid nanoparticle and its preparation method. Background Technology

[0002] In the biomedical field, protein therapy, as one of the most common techniques, plays a crucial role in areas such as vaccines and antibody therapy. Nanoparticles, due to their superior stability, higher density of carried antigenic epitopes, and ease of transport into cells, hold great promise for applications in protein therapy. However, the design limitations of the proteins carried by nanoparticles used in current protein drug formulations are significant, often requiring the introduction of metal ions and other non-biological materials, which can potentially lead to substantial side effects.

[0003] Meanwhile, nucleic acid drugs are shining brightly in cancer treatment, vaccines, and gene editing due to their short development cycle, broader target range, and ability to cure genetic diseases. To safely and accurately deliver nucleic acids into specific cells, nucleic acid transport vectors are typically used. However, existing nucleic acid transport vectors, such as lipid nanoparticles, have significant side effects, making the development of novel nucleic acid transport vectors an urgent priority. Summary of the Invention

[0004] The technical problem to be solved by this invention is to avoid the limitations of existing protein nanoparticles and the potential for significant side effects from the introduction of metal ions and other non-biological materials.

[0005] To address the above problems, the present invention provides a protein and nucleic acid nanoparticle.

[0006] The protein and nucleic acid nanoparticles provided by the present invention are nanoparticles made of nucleic acid molecules and protein molecules; the protein molecules contain peptides, the length of which is greater than or equal to 50 amino acid residues, the peptides contain greater than or equal to 12 positively charged amino acid residues, and the positively charged amino acid residues are arginine residues.

[0007] Furthermore, the diameter of the protein nucleic acid nanoparticles is 10nm-100nm.

[0008] Furthermore, the size of the nucleic acid molecule can be 21bp-4733bp. The size of the protein molecule can be 20768Da-50462Da.

[0009] Furthermore, the nucleic acid molecule is selected from at least one of the four types of nucleic acid molecules: single-stranded deoxyribonucleotide, double-stranded deoxyribonucleotide, single-stranded ribonucleotide, and double-stranded ribonucleotide.

[0010] The above-mentioned protein and nucleic acid nanoparticles are prepared according to any of the following methods.

[0011] This invention provides a method for preparing the above-mentioned protein-nucleic acid nanoparticles, comprising reacting the nucleic acid molecules and the protein molecules in a high ionic strength buffer solution, and reacting the resulting reaction product in a low ionic strength buffer solution to obtain the protein-nucleic acid nanoparticles; wherein the high ionic strength buffer solution contains 500 mM NaCl, 20 mM Tris-HCl, pH 8.0 or 2000 mM NaCl, 20 mM Tris-HCl, pH 8.0, wherein the NaCl content in the high ionic strength buffer solution is greater than or equal to 500 mM, and the low ionic strength buffer solution contains 100 mM NaCl, 20 mM Tris-HCl, pH 8.0, wherein the NaCl content in the low ionic strength buffer solution is less than or equal to 100 mM.

[0012] The NaCl content is greater than or equal to 500mM, which can be 500-2000mM, and less than or equal to 100mM, which can be 0-100mM.

[0013] In the above method, the mass ratio of the nucleic acid molecule to the protein molecule (based on the mass of PRM1 in the protein) is 1:0.3-3.3.

[0014] The concentration of the nucleic acid molecules in the high ionic strength buffer solution can be 20 ng / μL to 4 μg / μL.

[0015] In the above method, the 500mM NaCl, 20mM Tris-HCl, pH 8.0 or 2000mM NaCl, 20mM Tris-HCl, pH 8.0 high ionic strength buffer solution is composed of a solute and a solvent. The solvent is water, and the solute is NaCl or KCl or ammonium sulfate or sodium hydrogen phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate or potassium phosphate or dipotassium hydrogen phosphate or potassium dihydrogen phosphate, Tris-HCl or HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) or boric acid or calcium hydroxide or MOPS (3-morpholinopropanesulfonic acid) or CAPS (3-(cyclohexylamine)-1-propanesulfonic acid) or CHES (2-cyclohexylaminoethanesulfonic acid) or Tricine (tris(hydroxymethyl)methylglycine) or triethanolamine or sodium barbital or sodium carbonate.

[0016] The low ionic strength buffer solution consists of a solute and a solvent, wherein the solvent is water, and the solute is NaCl or KCl or ammonium sulfate or sodium hydrogen phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate or potassium phosphate or dipotassium hydrogen phosphate or potassium dihydrogen phosphate, Tris-HCl or HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) or boric acid or calcium hydroxide or MOPS (3-morpholinopropanesulfonic acid) or CAPS (3-(cyclohexylamine)-1-propanesulfonic acid) or CHES (2-cyclohexylaminoethanesulfonic acid) or Tricine (tris(hydroxymethyl)methylglycine) or triethanolamine or sodium barbital or sodium carbonate.

[0017] In the above method, the reaction in the low ionic strength buffer solution includes reacting the reaction product first in the high ionic strength buffer solution with a NaCl content of 500-2000 mM, and then reacting it in the low ionic strength buffer solution with a NaCl content of 0-100 mM.

[0018] In the above method, the protein-nucleic acid nanoparticles are nanoparticles made of nucleic acid molecules and protein molecules; the nucleic acid molecules are selected from at least one of the four types of nucleic acid molecules: single-stranded deoxyribonucleotides, double-stranded deoxyribonucleotides, single-stranded ribonucleotides, and double-stranded ribonucleotides.

[0019] In the above method, the protein molecule contains a peptide segment, the peptide segment having a length of 50 or more amino acid residues, and the peptide segment containing 12 or more positively charged arginine residues.

[0020] The protein may be any one of the following: A), B), C), (D), or (E):

[0021] The protein A) can be any of the following:

[0022] A1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0023] A2) A fusion protein obtained by attaching a fusion protein tag to the amino terminus of the protein shown in A1).

[0024] A3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues of proteins A1) or A2) have more than 80% identity with and the same function as proteins A1) or A2).

[0025] The protein described in B) can be any of the following:

[0026] B1) The amino acid sequence of this protein is that of SEQ ID No. 2;

[0027] B2) A fusion protein obtained by attaching a fusion protein tag to the amino terminus of the protein shown in B1).

[0028] B3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues of proteins B1) or B2) have more than 80% identity with and the same function as proteins B1) or B2).

[0029] The protein described in C) can be any of the following:

[0030] C1) The amino acid sequence is that of the protein in SEQ ID No. 3;

[0031] C2) The fusion protein obtained by attaching a fusion protein tag to the amino terminus of the protein shown in C1).

[0032] C3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues to proteins of C1) or C2) have more than 80% identity with and the same function as proteins of C1) or C2).

[0033] The protein described in D) can be any of the following:

[0034] D1) The amino acid sequence is that of the protein in SEQ ID No. 4;

[0035] D2) The fusion protein obtained by attaching a fusion protein tag to the amino terminus of the protein shown in D1).

[0036] D3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues to proteins of D1) or D2) have more than 80% identity with and the same function as proteins of D1) or D2).

[0037] The E) protein may be any of the following:

[0038] E1) The amino acid sequence of this protein is that of SEQ ID No. 5;

[0039] E2) is a fusion protein obtained by attaching a fusion protein tag to the amino terminus of the protein shown in E1).

[0040] E3) A protein that has more than 80% identity with and has the same function as the protein of E1) or E2) by substituting and / or deleting and / or adding amino acid residues.

[0041] This invention also provides the application of the above-mentioned protein and nucleic acid nanoparticles in the preparation of products that recognize tumor cells.

[0042] The present invention also provides the application of the above-mentioned protein and nucleic acid nanoparticles in the preparation of products that deliver proteins or nucleic acids or proteins and nucleic acids into cells.

[0043] The present invention provides a method for preparing nucleic acid-protein nanoparticles, which assembles proteins and nucleic acids using appropriate methods to ultimately form uniform nanoparticles with a diameter of approximately 30 nm. The proteins and nucleic acids in these particles possess significant engineering potential. Furthermore, since both the proteins and nucleic acids used are derived from biological sources, and the materials used in the assembly process are primarily inorganic substances abundant in organisms, such as sodium chloride, these particles exhibit good biocompatibility and can deliver proteins and nucleic acids into cells. This allows for applications in fields involving the use of proteins for treatment or prevention, such as vaccines and antibodies, as well as in fields using nucleic acids for treatment or prevention, such as nucleic acid vaccines, gene editing, and cancer treatment. Attached Figure Description

[0044] Figure 1 The particles obtained by assembly method 1 are shown. (A) Chromatogram of molecular sieve; (B) SDS-PAGE result after Coomassie Brilliant Blue staining; (C) Agarose gel result after GelRed staining; L: load, i.e., sample not purified by molecular sieve; M: marker; (D) Electron microscopy result of Peak 1 sample after negative staining, particles are within the green circle, scale bar: 100nm.

[0045] Figure 2 The particles obtained through assembly method 2 are shown. (A) shows the results of GelRed staining of the native agarose gel after assembly using a modified dialysis method with different protein-to-nucleic acid ratios; M: marker. (B) shows particles assembled using a modified dialysis method with a 1:3 nucleic acid-to-protein ratio; particles are shown within the green circle, and the scale bar is 100 nm. The ratio used is the mass ratio of DNA to protein during assembly.

[0046] Figure 3 These are particles formed by the assembly of different amino-terminal proteins and different nucleic acids.

[0047] Figure 4 These are particles formed by the mutation of the carboxyl-terminal protein and nucleic acid.

[0048] Figure 5The images show the results of particle digestion with different types of enzymes. (A) SDS-PAGE of particles digested with ULP1, stained with Coomassie Brilliant Blue, M: marker; (b) UREA-PAGE of particles digested with MNase, stained with GelRed, C: control, i.e., the individual 59nt ssDNA, M: marker; (c) Negative staining electron microscopy results of particles digested with ULP1; (d) Negative staining electron microscopy results of particles; (e) Negative staining electron microscopy results of particles digested with MNase. In the negative staining electron microscopy results, the green circles represent particles. Scale bar: 100nm.

[0049] Figure 6 Cellular uptake assays for HEK 293T cells. Nucleic acid-protein nanoparticles formed from SUMO-PRM1 were used. (A) Results observed 2 hours after particle addition, stained with Hoechst dye; (B) Results observed 4 hours after particle addition, stained with Hoechst dye; (C) Results observed 6 hours after particle addition, stained with Hoechst dye; (D) Results observed 6 hours after adding an equal amount of DNA alone, stained with Hoechst dye; (E) Results observed 6 hours after adding protein and DNA directly to cells under low-salt conditions in the same proportion used during assembly, stained with Hoechst dye. Merge: Fusion of images from bright field, 488nm excitation, and 346nm excitation. 488: Image under 488nm excitation. Hoechst: Image under 346nm excitation. Scale bars in the figures are 50μm.

[0050] Figure 7Cellular uptake experiment for HeLa cells. A. After adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 to HeLa cells and incubating for 2 hours, the cells were stained with Hoechst dye and observed. B. After adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to HeLa cells and incubating for 2 hours, the cells were stained with Hoechst dye and observed. C. After adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 to HeLa cells and incubating for 4 hours, the cells were stained with Hoechst dye and observed. D. After adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to HeLa cells and incubating for 4 hours, the cells were stained with Hoechst dye and observed. E. After adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 to HeLa cells and incubating for 6 hours, the cells were stained with Hoechst dye and observed. F. After adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to HeLa cells and incubating for 6 hours, the cells were stained with Hoechst dye and observed. G. After adding an equal amount of DNA to HeLa cells and incubating for 6 hours, the cells were stained with Hoechst dye and observed. Merge: A fusion of images of the bright field, 488nm excitation, and 346nm excitation. 488: Image under 488nm excitation. Hoechest: Image under 346nm excitation. The scale bar in the figures is 50μm.

[0051] Figure 8 Cellular uptake assay for HEK 293T cells. A. Observation 8 hours after adding nucleic acid-protein nanoparticles to HEK 293T cells. B. Observation 28 hours after adding nucleic acid-protein nanoparticles to HEK 293T cells. Detailed Implementation

[0052] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0054] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0055] The carrier pET-28a used in this invention is described in the following literature: Zhang L, Serra-Cardona A, Zhou H, et al. Multisite Substrate Recognition in Asf1-Dependent Acetylation of Histone H3 K56 by Rtt109. Cell, 2018, 174(4):818-830. It is available to the public from the Institute of Biophysics, Chinese Academy of Sciences. This biomaterial is for the purpose of repeating experiments of this invention only and should not be used for other purposes.

[0056] The plasmids pBluescriptII, pET-22b, BL21-CondonPlus-RIL E. coli competent cells, and DH5αE. coli competent cells used in this invention have been described in: Yang D, Fang Q, Wang M, et al. Nα-acetylated Sir3 stabilizes the conformation of a nucleosome-binding loop in the BAH domain. Nature Structural & Molecular Biology, 2013, 20(9):1116-1118. These materials are available to the public from the Institute of Biophysics, Chinese Academy of Sciences. This biomaterial is intended solely for repeating experiments of this invention and should not be used for any other purpose.

[0057] The HEK 293T cells and plasmid pGEX-6P-1 used in this invention have been described in: Liu CP, Xiong C, Wang M, et al. Structure of the variant histone H3.3-H4 heterodimer in complex with its chaperone DAXX. Nat Struct Mol Biol, 2012, 19(12):1287-1292. These materials are available to the public from the Institute of Biophysics, Chinese Academy of Sciences. This biological material is intended solely for repeating experiments of this invention and should not be used for any other purpose.

[0058] The plasmid pEGFP-N1 used in this invention has been described in: Liu CP, Jin W, Hu J, et al. Distincthistone H3-H4 binding modes of sNASP reveal the basis for cooperation and competition of histone chaperones. Genes Dev, 2021, 35(23-24):1610-1624. It is publicly available from the Institute of Biophysics, Chinese Academy of Sciences. This biological material is solely for the purpose of replicating experiments of this invention and should not be used for any other purpose.

[0059] The HeLa cells used in this invention have been described in: Fang J, Liu Y, Wei Y, et al. Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N. Genes Dev, 2015, 29(10):1058-1073. They are available to the public from the Institute of Biophysics, Chinese Academy of Sciences. This biological material is solely for the purpose of replicating experiments of this invention and should not be used for any other purpose.

[0060] The pri-miRNA used in this invention is described in: Jin W, Wang J, Liu CP, et al. Structural Basis for pri-miRNA Recognition by Drosha. Molecular Cell, 2020, 78(3):423-433. It is available to the public from the Institute of Biophysics, Chinese Academy of Sciences. This biological material is for the sole purpose of repeating experiments of this invention and should not be used for any other purpose.

[0061] ULP1 in this invention is described in: Hou P, Huang C, Liu CP, et al. Structural Insights into Stimulation of Ash1L's H3K36 Methyltransferase Activity through Mrg15 Binding. Structure, 2019, 27(5):837-845. It is available to the public from the Institute of Biophysics, Chinese Academy of Sciences. This biomaterial is for the purpose of repeating experiments of this invention only and should not be used for other purposes.

[0062] The proteins selected in this study are SUMO-PRM1, GST-PRM1, and MBP-PRM1. The specific amino acid sequences of the proteins are shown in Table 1, and the nucleotide sequences of the proteins are shown in Table 3.

[0063] The preparation methods for proteins SUMO-PRM1, GST-PRM1, MBP-PRM1, SUMO-PRM1(mut), and GE11-SUMO-PRM1 are as follows:

[0064] 1. Using a prokaryotic expression system to express proteins

[0065] The pET-28a gene expression vector containing the SUMO-PRM1 gene (see SEQ 13 for the specific sequence), the pET-22b gene expression vector containing the MBP-PRM1 gene (see SEQ 14 for the specific sequence), the pGEX-6P-1 gene expression vector containing the GST-PRM1 gene (see SEQ 15 for the specific sequence), the pET-28a gene expression vector containing the SUMO-PRM1(mut) gene (see SEQ 16 for the specific sequence), and the pET-28a gene expression vector containing the GE11-SUMO-PRM1 gene (see SEQ 17 for the specific sequence) were used respectively.

[0066] 1) Transformation: Take 50 ng of expression plasmid (SUMO-PRM1 gene expression vector, SUMO-PRM1(mut) gene expression vector, GE11-SUMO-PRM1 gene expression vector, GST-PRM1 gene expression vector, or MBP-PRM1 gene expression vector) and add it to 50 μL of BL21-CondonPlus-RIL in advance. In E. coli competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, add 800 μL of culture medium, and incubate at 37℃ and 180 rpm for 1 h in a shaker. Take 200 μL of bacterial culture and transfer it to LB solid medium supplemented with the corresponding antibiotics (50 μg / mL kanamycin and 37 μg / mL chloramphenicol for SUMO-PRM1 gene expression vector, SUMO-PRM1(mut) gene expression vector, or GE11-SUMO-PRM1 gene expression vector; 50 μg / mL ampicillin and 37 μg / mL chloramphenicol for GST-PRM1 gene expression vector or MBP-PRM1 gene expression vector), and incubate at 37℃ for 12 h.

[0067] 2) Bacterial culture: Pick a single colony and add it to 5 mL of LB medium containing the corresponding antibiotics (50 μg / mL kanamycin and 37 μg / mL chloramphenicol for SUMO-PRM1 gene expression vector, SUMO-PRM1(mut) gene expression vector or GE11-SUMO-PRM1 gene expression vector; 50 μg / mL ampicillin and 37 μg / mL chloramphenicol for GST-PRM1 gene expression vector or MBP-PRM1 gene expression vector). Incubate at 37°C and 180 rpm for 8 h in a shaker. Then add the antibiotics to 200 mL of LB medium containing the corresponding antibiotics at a ratio of 1:1000 and incubate at 37°C and 180 rpm for 12 h in a shaker.

[0068] 3) Scale-up culture: Add the cultured bacterial suspension to 800 mL of LB medium containing the appropriate antibiotic at a ratio of 1:40, and incubate at 160 rpm for about 4 hours in a shaker at 37°C until the OD of the bacterial suspension reaches its maximum value. 600 The value was approximately 0.6. At this point, IPTG was added to a final concentration of 0.5 mM, and the mixture was placed in a shaker at 37°C and incubated at 160 rpm for 4 hours.

[0069] 4) Collect bacterial culture: Transfer each bottle of bacterial culture to a 1L centrifuge bottle. Centrifuge at 4000rpm for 15 minutes using a floor centrifuge at 4℃ to collect the bacterial cells and discard the supernatant.

[0070] 5) Resuspend the bacterial cells by adding 200 mL of cell lysis buffer per 10 L of bacterial culture.

[0071] 2. Protein purification

[0072] 1) Sterilization

[0073] The resuspended bacterial solution was disrupted by ultrasonic or high-pressure disruption. The disrupted bacterial solution was then centrifuged at 4°C, 20000g for 60 minutes. The supernatant was retained and the precipitate was discarded.

[0074] 2) Affinity chromatography

[0075] The supernatant after centrifugation was incubated with the corresponding chromatography medium at 4°C. Ni-excelbeads (used for purifying SUMO-PRM1, SUMO-PRM1(mut), and GE11-SUMO-PRM1) were incubated for 30 min, while GSTbeads (used for purifying GST-PRM1) and Amylose Resin beads (used for purifying MBP-PRM1) were incubated for 3 h.

[0076] The supernatant was flow-through using a gravity column, followed by washing the chromatography medium with 10 column volumes of cell lysis buffer to remove non-specifically bound contaminating proteins.

[0077] The chromatography medium is eluted with the appropriate eluent, and the eluent is collected.

[0078] 3) Ion exchange chromatography

[0079] The ion column used was a heparin column (purchased from GE Healthcare, catalog number 17040703).

[0080] a. Wash the ion column with 10 column volumes of ion column buffer A.

[0081] b. Desalt the affinity chromatography buffer to the salt concentration of ion column buffer A and flow it through the ion column.

[0082] c. Wash the ion column again using ion column buffer A.

[0083] d. Set the AKTA system program to elute the ion column by increasing the concentration of ion column buffer B in the ion column flow-through solution from 0 to 100% over 50 minutes at a flow rate of 2 mL / min.

[0084] e. Collect the corresponding samples based on the chromatograms from the AKTA system, and collect the corresponding samples based on the Coomassie Brilliant Blue staining results from SDS-PAGE.

[0085] f. Wash 10 column volumes again using ion column buffer B.

[0086] g. Use water purified by Millipore, filter it, and then clean the system and the ion column.

[0087] 4) Size exclusion chromatography

[0088] a. Wash the HiLoad 16 / 600 Superdex 75pg molecular sieve (GE Healthcare, catalog number 28989333) with molecular sieve buffer A for 1.5 column volumes until the salt concentration of the molecular sieve effluent is the same as that of the molecular sieve buffer.

[0089] b. Concentrate the sample collected by the ion column, centrifuge at 4°C, 20000g for 30 min, and load the sample into the molecular sieve using the loading loop.

[0090] 3. Based on the chromatogram of the AKTA system and the Coomassie Brilliant Blue staining results of SDS-PAGE, the corresponding samples were collected to obtain proteins SUMO-PRM1, GST-PRM1 and MBP-PRM1 respectively.

[0091] The selected nucleic acids were: 59nt ssDNA, 59bp DNA, pEGFP-N1 linearized product, 6×187bp DNA, siRNA, pri-miRNA, EGFP mRNA, and 94bp DNA.

[0092] The following are methods for preparing large quantities of DNA fragments (taking 94bp DNA and 6×187bp DNA as examples):

[0093] 1. Mass replication of plasmids

[0094] 94 bp DNA (nucleotide sequence see SEQ ID No. 10) and 6 × 187 bp DNA (nucleotide sequence see SEQ ID No. 11) were inserted into the multiple cloning site of pBluescriptII to obtain recombinant vectors containing 94 bp DNA and 6 × 187 bp DNA, respectively.

[0095] 1) Transformation: Take 50 ng of any of the above recombinant vectors and add it to 50 μL of DH5αE.coli competent cells. Incubate on ice for 30 min, heat shock at 42℃ for 90 s, add 800 μL of culture medium, and incubate at 37℃ and 180 rpm for 1 h on a shaker. Take 200 μL of bacterial culture and transfer it to LB solid medium supplemented with the corresponding antibiotic (50 μg / mL ampicillin for pBluescriptII plasmid and 50 μg / mL kanamycin for pEGFP-N1). Incubate at 37℃ for 12 h.

[0096] 2) Add the cultured bacterial suspension to 800 mL of TB medium containing the appropriate antibiotics (50 μg / mL ampicillin for pBluescriptII plasmid, and 50 μg / mL kanamycin for pEGFP-N1) and phosphate buffer at a ratio of 1:40. Incubate at 160 rpm for approximately 6 hours at 37°C until the OD of the bacterial suspension reaches its maximum value. 600 The value was approximately 0.8. The temperature of the shaker was increased to 42℃, and the culture was continued at 160 rpm for 12 hours.

[0097] 3) Collect the bacterial culture. Transfer each bottle of bacterial culture to a 1L centrifuge bottle. Centrifuge at 4°C and 4000rpm for 15 minutes using a floor centrifuge to collect the bacterial cells and discard the supernatant.

[0098] 2. Plasmid extraction by alkaline lysis method

[0099] 1) Solution preparation

[0100] S1: Weigh 15mL Tris-HCl (pH 8.0), 5.93g D-glucose, and 12mL EDTA (pH 8.0), and add water to a final volume of 500mL. Prepare fresh before use.

[0101] S2: Weigh 6.4g NaOH and 8g SDS, add water to make up to 800mL, and prepare fresh before use.

[0102] S3: Weigh 588.8g of potassium acetate and 229.3mL of glacial acetic acid, add water to make up to 2L and then filter.

[0103] 40% PEG6000 solution: Weigh 20g of PEG6000 into 40mL of water, heat in a 65℃ water bath to dissolve, and then dilute to 50mL for later use.

[0104] TE: 10mM Tris-HCl pH 8.0, 1mM EDTA.

[0105] TE 10 / 0.1: 10mM Tris-HCl pH 8.0, 0.1mM EDTA.

[0106] TE 10 / 50: 10mM Tris-HCl pH 8.0, 50mM EDTA.

[0107] 3M sodium acetate solution: Weigh 40.8g CH3COONa·H2O, add about 40mL of water and stir to dissolve, add glacial acetic acid to adjust the pH to 5.2, add water to make up to 100mL, and store at 4℃ for later use.

[0108] Phenol-chloroform extract: Mix Tris-saturated phenol and chloroform at a volume ratio of 1:1, retain the aqueous phase, and let stand for later use.

[0109] Chloroform-isoamyl alcohol extract: Mix isoamyl alcohol and chloroform at a volume ratio of 1:24 and let stand for later use.

[0110] 2) Mix 10L of bacterial culture with 360mL of plasmid extraction buffer S1, and resuspend the bacterial cells in a shaker at 37℃ and 160rpm for about 30min until no bacterial clumps are visible to the naked eye.

[0111] 3) Add fresh plasmid extraction buffer S2 at a volume ratio of S1:S2 = 1:2, and gently invert to mix thoroughly. Let stand at room temperature for 5 minutes.

[0112] 4) Add the pre-cooled plasmid extraction buffer S3 according to the volume ratio of S1:S3 = 1:3.5, and shake until there is no layering and the impurities are in the form of egg drop soup.

[0113] 5) Place on ice for 10 minutes. Centrifuge at 4000 rpm and 4°C for 30 minutes using a floor centrifuge. Carefully aspirate the supernatant, filter it through 4 layers of gauze to remove suspended impurities, and transfer it to a clean beaker.

[0114] 6) Isopropanol precipitation of DNA: Add 0.52 volumes of isopropanol to the collected supernatant, mix thoroughly, and incubate at room temperature until a clear precipitate forms and separates from the liquid phase. Centrifuge at 4000 rpm for 30 min using a floor centrifuge, discard the supernatant, and recover the nucleic acid precipitate. Gently rinse the walls and bottom of the centrifuge tube with a small amount of 70% ethanol to remove the precipitate, and allow the remaining ethanol to evaporate at room temperature. Then add 50 mL of TE 10 / 50 to dissolve the precipitate.

[0115] 7) RNase digestion: Add an appropriate amount of RNase to the recovered plasmid and digest overnight at 37°C to completely shred the RNA in the plasmid.

[0116] 8) Phenol-chloroform protein removal: Prepare phenol-chloroform in advance and let it stand until clear phase separation occurs. After RNase digestion, add 1 / 5 volume of phenol-chloroform, vortex to mix, centrifuge at 20℃, 20000g for 20min, carefully aspirate the upper aqueous phase and retain it. Repeat this process until the interface between the two phases is clear and no white precipitate is formed.

[0117] 9) Chloroform-isoamyl alcohol for phenol removal: Then add 2 / 5 volume of chloroform-isoamyl alcohol, shake to mix, centrifuge at 20℃, 20000g for 20min, and carefully aspirate the upper aqueous phase for retention.

[0118] 10) RNA removal with PEG: Add 1 / 5 volume of 4M NaCl and 2 / 5 volume of 40% PEG6000 to the aspirated aqueous phase, mix well, and incubate at 37°C for 5 min, then incubate on ice for 30 min. Centrifuge at 20000g for 15 min at 4°C, discard the supernatant (containing RNA), wash the tube wall and precipitate with a small amount of 70% ethanol without disturbing the precipitate, discard the ethanol, and dry with a hairdryer. Dissolve the precipitate in 30 mL of TE10 / 0.1 and incubate at 37°C for several hours until the precipitate is completely dissolved.

[0119] 11) Remove PEG: Add 1 / 5 volume of chloroform isoamyl alcohol, shake to mix, centrifuge at 20000g for 10 min at 4℃, repeat until there is no obvious precipitate after centrifugation.

[0120] 12) Collect plasmids by ethanol precipitation: Add 1 / 10 volume of 3M sodium acetate solution (pH 5.2) and 2.5 volumes of ice-cold anhydrous ethanol, and incubate at -20°C for at least 1 h. Centrifuge at 20,000 g for 15 min at 4°C to precipitate plasmid DNA. Rinse the container walls and the surface of the precipitate with a small amount of 70% ethanol, discard the ethanol, and dry with a hairdryer. Dissolve in 30 mL of TE10 / 0.1.

[0121] 3. Obtaining DNA fragments through enzyme digestion

[0122] 1) Enzyme digestion: Add the corresponding enzyme at a ratio of 30 U per 1 mg of plasmid (use AseI for linearized pEGFP-N1 plasmid, and EcoV for others), and add DTT to a final concentration of 1 mM.

[0123] 2) Fragment separation (this step is unnecessary for linearized plasmid products): Add 0.192 volumes of 4 M NaCl and the corresponding volume of 40% PEG6000, incubate at 37°C for 5 min, then incubate on ice for 1 h (do not incubate on ice for too long to avoid some of the target fragments also settling), centrifuge at 4°C, 20000 g for 20 min, and the precipitate is plasmid DNA. The specific PEG volume needs to be tested in small batches using the same proportion beforehand until the supernatant has no large fragments and the precipitate has almost no small fragments.

[0124] 3) Ethanol precipitation and product collection: Add 1 / 10 volume of ice-cold 3 M NaAc (pH 5.2) and 2.5 volumes of ice-cold anhydrous ethanol, and incubate at -20°C for at least 1 h. Centrifuge at 20000 g for 15 min at 4°C. The precipitate contains the target plasmid DNA. Rinse the container walls and precipitate surface with a small amount of 70% ethanol, discard the ethanol, and dry with a hairdryer. Dissolve in 30 mL TE 10 / 0.1. This yields the pEGFP-N1 linearized product, 6 × 187 bp DNA, and 94 bp DNA, respectively.

[0125] 4. The 59 nt ssDNA is a custom product of Sangon Biotech (Shanghai) Co., Ltd., with order number 111930348.

[0126] 5. The 59 bp DNA was obtained by annealing 59 nt ssDNA using a PCR instrument (BIO-RAD, catalog number: 1851148). Annealing process: The 59 nt ssDNA was dissolved in water at a concentration of 1 mg / mL. The sample was heated to 100°C and held for 5 minutes. Then, the temperature was decreased by 0.5°C every 30 seconds until it reached 20°C.

[0127] 6. The nucleotide sequence of siRNA is shown in Table 2. It is a customized product of Sangon Biotech (Shanghai) Co., Ltd., order number: R 12444.

[0128] The preparation method of Tris-HCl used in this invention is as follows:

[0129] First, prepare a 1 M Tris-HCl stock solution with pH 8.0: Weigh 121.1 g Tris (purchased from Sigma-Aldrich, catalog number: V900483), dissolve in 800 mL of water, ensure complete dissolution, adjust the pH to 8.0 with hydrochloric acid, and bring the volume to 1 L. When using, dilute the stock solution to the appropriate concentration.

[0130] In this invention, the buffer solution used during particle assembly is water as the solvent, and the solute can be NaCl, KCl, ammonium sulfate, sodium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, Tris-HCl, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), boric acid, calcium hydroxide, MOPS (3-morpholinopropanesulfonic acid), CAPS (3-(cyclohexylamine)-1-propanesulfonic acid), CHES (2-cyclohexylaminoethanesulfonic acid), Tricine (tris(hydroxymethyl)methylglycine), triethanolamine, sodium barbital, or sodium carbonate. The solutes used in the following examples are NaCl and Tris-HCl.

[0131] Example 1: Assembly method of nucleic acid protein nanoparticles:

[0132] The preparation method for buffer A is as follows: 2000 mM NaCl, 20 mM Tris-HCl, pH 8.0;

[0133] The preparation method for buffer B is as follows: 500mM NaCl, 20mM Tris-HCl, pH 8.0;

[0134] The preparation method for buffer C is as follows: 100mM NaCl, 20mM Tris-HCl, pH 8.0

[0135] 1. Assembly Method 1

[0136] The following example illustrates the preparation method of protein-nucleic acid nanoparticles (Peak 1 sample) using a protein named SUMO-PRM1 (amino acid sequence SEQ ID No. 1) as the protein molecule and a 94bp double-stranded DNA molecule (nucleotide sequence SEQ ID No. 10, abbreviated as 94bp DNA) as the nucleic acid molecule:

[0137] ① Buffer A, Buffer B, and Buffer C required for precooling at 4℃;

[0138] ② At 4℃, add SUMO-PRM1 and 94bp DNA to buffer A to obtain reaction solution A. The volume of reaction solution A is 0.5mL, the concentration of SUMO-PRM1 is 4mg / mL, and the concentration of 94bp DNA is 4mg / mL. Place reaction solution A into a dialysis tube (MilliporeSigma, catalog number 71509-3) with a molecular weight cutoff of 6-8kDa, place the dialysis tube in buffer A, and place it on a magnetic stirrer (Chlimbell, catalog number 81-2). Dialyze at 4℃ for 3h.

[0139] ③ Take out the dialysis tube, place it in buffer B, put it on a magnetic stirrer, and dialyze at 4°C for 3 hours;

[0140] ④ Remove the dialysis tube, place it in buffer C, and put it on a magnetic stirrer. Dialyze at 4°C for 3 hours to obtain protein-nucleic acid nanoparticles, which are named SUMO-PRM1-94 bp DNA. Remove the sample for subsequent experiments.

[0141] After assembly, the sample was ultrafiltered to approximately 0.4 mL using a 30 kDa ultrafiltration tube (MilliporeSigma, catalog number: UFC903096) at 3000 g and 4°C. The sample was then centrifuged at 20000 g and 4°C for 15 minutes before being loaded onto a Superose 6 (GE Healthcare, catalog number: 29-0915-96) gel filtration chromatography system. The eluent was 100 mM NaCl and 20 mM Tris-HCl solution, with a loading volume of 0.4 mL and an elution rate of 0.5 mL / min. Figure A shows the chromatogram displayed by the protein purification system used (GE Healthcare, model AKTA Pure25M1). The eluted samples at Peak 1 and Peak 2 in Figure A were subjected to polyacrylamide gel electrophoresis to obtain Figure B, and then subjected to agarose gel electrophoresis under denaturing conditions to obtain Figure C. Simultaneously, the eluted sample at Peak 1 was negatively stained and observed under an electron microscope to obtain Figure D.

[0142] Depend on Figure 1 The chromatogram of the medium molecular sieve shows that the first sample peak appeared at a position of about 0.5 column volumes (about 12 mL), which was named Peak 1, and the second sample peak appeared at a position of about 0.625 column volumes (15 mL), which was named Peak 2.

[0143] SDS-PAGE analysis of the nucleic acid-protein nanoparticles obtained in the above steps yielded the following results: Figure 1 As shown in Figure B, both Peak 1 and Peak 2 contain SUMO-PRM1, with the protein mainly concentrated in Peak 1.

[0144] The results of agarose gel staining with Gel-Red are as follows Figure 1 As shown in Figure C, both Peak 1 and Peak 2 contain 94 bp of DNA.

[0145] Peak 1 sample negative staining electron microscopy examination ( Figure 1 In the study, it was found that the Peak 1 sample contained uniformly sized particles with a diameter of approximately 30 nm.

[0146] 2. Assembly Method 2

[0147] SUMO-PRM1 (SEQ ID No. 1) and 94bp DNA (SEQ ID No. 10) were used to obtain protein-nucleic acid nanoparticles through the following steps:

[0148] ① Buffer solution C required for precooling at 4℃.

[0149] ② Under buffer A conditions, the protein SUMO-PRM1 and nucleic acid (shown in SEQ ID No. 10) were mixed in the following proportions: the mass ratio of nucleic acid to protein SUMO-PRM1 was 1:1, 1:2, and 1:3. The mixture was then added to a dialysis tube (MilliporeSigma, catalog number 71509-3), placed in buffer C, and dialyzed on a magnetic stirrer for 3 hours. The sample was then removed for subsequent experiments.

[0150] The particles obtained from assembly method 2 were stained with Gel-Red and subjected to agarose gel electrophoresis under non-denaturing conditions. The results are as follows: Figure 2 As shown in Figure A, as the proportion of protein added during assembly increases, the degree to which the assembled product migrates upward on the agarose gel also increases accordingly, indicating that the protein binds to the nucleic acid, and the binding strength also increases with the increase of the proportion of protein during assembly. Figure 2 As shown in Figure B, particles assembled using a modified dialysis method with a mass ratio of 1:3 nucleic acid and protein appear as particles with a diameter of approximately 30 nm under an electron microscope.

[0151] Electron microscopy results showed that both assembly method 1 and assembly method 2 could produce particles with a diameter of approximately 30 nm under an electron microscope. Assembly method 1 produced particles with better uniformity. However, assembly method 2 was simpler and required less time.

[0152] Example 2: Investigating the effects of different proteins and nucleic acids on the formation of protein-nucleic acid nanoparticles.

[0153] (1) Comparison of proteins and nucleic acids

[0154] Different types of proteins (specific protein sequences are shown in Table 1) are assembled with different types of nucleic acids (specific sequences are shown in Table 2).

[0155] The selected proteins are SUMO-PRM1, GST-PRM1, and MBP-PRM1. The nucleotide sequences of each protein are shown in Table 3.

[0156] The selected nucleic acids were 59nt ssDNA, 59bp DNA, pEGFP-N1 linearized product, 6×187bp DNA, siRNA, pri-miRNA, EGFP mRNA, and 94bp DNA. Specific nucleic acid sequences are shown in Table 2.

[0157] The amino acid sequences of the proteins used in Table 1

[0158]

[0159]

[0160] The sequences of nucleic acids used in Table 2

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] Note: EGFP mRNA is a 996nt mRNA from APExBio, catalog number R1001. Conventionally synthesized siRNAs are 21-25nt long, double-stranded small RNAs with a 2nt dT / UU dT hanger at the 3' end. The bioengineered double-stranded siRNA (i.e., the siRNA used in this patent) has the following characteristics: the sense strand consists of a 19-base target sequence plus a 2-base TT hanger at the 3' end, with the sequence 5'-CAAGCUGACCCUGAAGUUCTT-3'; the antisense strand consists of 19 complementary bases to the sense strand, plus a 2-base TT hanger at the 3' end, with the sequence 5'-GAACUUCAGGGUCAGCUUGTT-3'.

[0167] The nucleotide sequences of the proteins used in Table 3

[0168]

[0169]

[0170]

[0171]

[0172] These proteins involved in assembly are characterized by having 12 arginine residues out of their 50 amino acids, while nucleic acids are not significantly different.

[0173] The specific assembly method and proportions (mass ratio) of the nucleic acid protein nanoparticles are as follows:

[0174] 1) 59nt ssDNA: SUMO-PRM1 = 1:3, where the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1;

[0175] 2) 59bp DNA: SUMO-PRM1 = 1:3, where the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1;

[0176] 3) 6×187bp DNA: SUMO-PRM1 = 1:6, where the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1;

[0177] 4) pEGFP-N1 linearized product: SUMO-PRM1 = 1:5, wherein the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1;

[0178] 5) siRNA: SUMO-PRM1 = 1:10, where the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1;

[0179] 6) pri-miRNA: SUMO-PRM1 = 1:2, where the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1;

[0180] 7) EGFP mRNA: SUMO-PRM1 = 1:4, where the mass ratio of SUMO to PRM1 in SUMO-PRM1 is 2:1;

[0181] 8) 59nt ssDNA: GST-PRM1 = 1:6, where the mass ratio of GST to PRM1 in GST-PRM1 is 3:1;

[0182] 9) 59bp DNA: GST-PRM1 = 1:6, where the mass ratio of GST to PRM1 in GST-PRM1 is 3:1;

[0183] 10) 94bp DNA: GST-PRM1 = 1:6, where the mass ratio of GST to PRM1 in GST-PRM1 is 3:1;

[0184] 11) pEGFP-N1 linearized product: GST-PRM1 = 1:4, wherein the mass ratio of GST to PRM1 in GST-PRM1 is 3:1;

[0185] 12) 59nt ssDNA: MBP-PRM1 = 1:15, where the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1;

[0186] 13) 59bp DNA: MBP-PRM1 = 1:15, where the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1;

[0187] 14) 94bp DNA: MBP-PRM1 = 1:15, where the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1;

[0188] 15) pEGFP-N1 linearized product: MBP-PRM1 = 1:15, wherein the mass ratio of MBP to PRM1 in MBP-PRM1 is 6:1.

[0189] The results are as follows Figure 3As shown, 59nt ssDNA can form particles with a diameter of approximately 30nm with SUMO-PRM1 through assembly mode 2; 59bp DNA can form particles with a diameter of approximately 30nm with SUMO-PRM1 through assembly mode 2; 6×187bp DNA can form particles with a diameter of approximately 30nm with SUMO-PRM1 through assembly mode 2; pEGFP-N1 linearized product can form particles with a diameter of approximately 30nm with SUMO-PRM1 through assembly mode 2; siRNA can form particles with a diameter of approximately 30nm with SUMO-PRM1 through assembly mode 2; per-miRNA can form particles with a diameter of approximately 30nm with SUMO-PRM1 through assembly mode 2; EGFP mRNA can form particles with a diameter of approximately 30nm with SUMO-PRM1 through assembly mode 2; 59nt ssDNA can form particles with a diameter of approximately 30nm with GST-PRM1 through assembly mode 2; 59bp DNA can form particles with a diameter of approximately 30nm with GST-PRM1 through assembly mode 2; 94bp DNA can form particles with a diameter of approximately 30 nm with GST-PRM1 through assembly mode 2; linearized pEGFP-N1 products can form particles with a diameter of approximately 30 nm with GST-PRM1 through assembly mode 2; 59 nt ssDNA can form particles with a diameter of approximately 30 nm with MBP-PRM1 through assembly mode 2; 59 bp DNA can form particles with a diameter of approximately 30 nm with MBP-PRM1 through assembly mode 2; 94 bp DNA can form particles with a diameter of approximately 30 nm with MBP-PRM1 through assembly mode 2; linearized pEGFP-N1 products can form particles with a diameter of approximately 30 nm with MBP-PRM1 through assembly mode 2.

[0190] In summary, SUMO-PRM1 can form particles with a diameter of approximately 30 nm with DNA or RNA of different lengths or sequences, and of different lengths or sequences, through assembly method 2. SUMO-PRM1, GST-PRM1, and MBP-PRM1 can also form particles with a diameter of approximately 30 nm with single-stranded or double-stranded DNA of different lengths or sequences.

[0191] This leads to the conclusion that after fusing other proteins to the N-terminus of PRM1, it can form particles with a diameter of approximately 30 nm with nucleic acids of different lengths and sequences through assembly mode 2.

[0192] (2) Effect of mutating half of the arginine residues in the PRM1 sequence of SUMO-PRM1 to alanine on the formation of nucleic acid-protein nanoparticles

[0193] Half of the arginine residues in the PRM1 sequence of SUMO-PRM1 were mutated to alanine (the specific sequence is SEQ ID No. 4). The mass ratio of the protein to the linearized product of the pEGFP plasmid was SUMO-PRM1(mut):nucleic acid = 4:1, of which the mass ratio of SUMO:PRM1(mut) was 2:1. The protein was then assembled according to assembly method 2.

[0194] Assembly results as follows Figure 4 As shown, based on assembly method 2, the mutated protein can still form particles with a diameter of about 30 nm with nucleic acids.

[0195] As shown above, if more than 12 arginine residues appear in any 50-amino acid sequence of a protein, the protein can be assembled into uniformly sized nanoparticles using the assembly method described in this paper.

[0196] The protein in the nanoparticles of this invention only requires the addition of a certain number of positively charged amino acids, such as arginine, to its sequence. By reducing the salt concentration in the buffer solution, it can assemble with deoxyribonucleotides or ribonucleotides of different lengths, single-stranded or double-stranded, and different sequences to form nanoparticles with a size of approximately 30 nm. The three-dimensional structure of the amino terminus of the protein in these nanoparticles can be fully recognized, thereby enabling them to perform their corresponding functions.

[0197] Example 3: Application of protein and nucleic acid nanoparticles

[0198] The protein-nucleic acid nanoparticles (SUMO-PRM1 and 59nt ssDNA) obtained in Example 1 were treated with MNase (Micrococcal nuclease, Thermo Fisher Scientific, catalog number 88216) and ULP1. The specific steps are as follows: The particles were assembled according to assembly method 2 and diluted with 100mM NaCl, 20mM Tris-HCl, and pH 8.0 buffer to a final concentration of 20 ng / μL (the result was obtained by measuring single-stranded DNA using a Nanodrop 2000 spectrophotometer). A: 1 μL of ULP1 (4 mg / mL) was added to every 50 μL of sample. After incubation on ice for 3 hours, 1 μL of ULP1, 5 μL of particles with added ULP1, and the untreated particles were analyzed by SDS-PAGE. B: Add MNase to the low-salt buffer containing particles and the low-salt buffer containing 20 ng / μL 59nt ssDNA at a ratio of 1 μL per 50 μL sample. After incubating on ice for 3 h, take the particle buffer with added MNase, the buffer with particles without added MNase, and the buffer with 59nt ssDNA for urea gel detection.

[0199] The efficient cleavage of SUMO by the ULP1 enzyme depends on its recognition of the three-dimensional sequence of SUMO. Figure 5 As can be seen, under these conditions, the protein in the particles is efficiently cleaved by ULP1. At the same time, under these conditions, the individual 59nt ssDNA is completely degraded by MNase, while the 59nt ssDNA assembled into nanoparticles does not show severe degradation.

[0200] Therefore, the protein-nucleic acid nanoparticles have a certain protective effect on nucleic acids. At the same time, the three-dimensional structure of the amino terminus of the protein can be recognized by other proteins and perform corresponding functions, indicating the corresponding ability of the particles in protein delivery.

[0201] Example 4: Cellular Experiment Results of Protein and Nucleic Acid Nanoparticles

[0202] 1. Cell uptake assay of HEK 293T cells (the nucleic acids used were fluorescently labeled nucleic acids).

[0203] The DNA molecule used in this experiment was DNA with Alexa flour 488 fluorescent modification at the 5' end (a custom product of Sangon Biotech (Shanghai) Co., Ltd., order number: 111989851, specific sequence see SEQ ID No. 8), and the protein used was SUMO-PRM1, specific sequence see SEQ ID No. 1. The mass ratio of SUMO-PRM1 protein to 59nt ssDNA was 4:1, and assembly was performed according to assembly method 2. The experimental groups are as follows:

[0204] (A) Two hours after adding nucleic acid-protein nanoparticles to HEK 293T cells, the cells were stained with Hoechst dye (purchased from Beyotime Biotechnology Co., Ltd., product code: C1022, see product instructions for specific usage) and observed.

[0205] (B) Observation after adding nucleic acid-protein nanoparticles to HEK 293T cells and staining with Hoechst dye 4 hours later;

[0206] (C) Observation after staining with Hoechst dye 6 hours after adding nucleic acid-protein nanoparticles to HEK 293T cells;

[0207] (D) Observation after adding an equal amount of DNA to HEK 293T cells alone and staining with Hoechst dye 6 hours later;

[0208] (E) The protein and DNA were mixed directly according to the ratio used for assembly, and observed after 6 hours in the cells.

[0209] Observations were conducted under the conditions of Merge, bright field, 488 nm wavelength excitation, and 346 nm wavelength excitation light. The results are as follows: Figure 6 As shown, compared to (D) where an equal amount of DNA was directly added, the results of (A), (B), and (C) indicate that nucleic acids were already being taken up by cells two hours after the addition of the particles, and the amount of nucleic acids taken up by cells increased over time. Furthermore, a comparison with the results of (E) leads to the conclusion that nucleic acid uptake by cells depends on the assembled 30nm particles. This demonstrates that the particles can be taken up by HEK 293T cells, thereby transporting nucleic acids into the cells.

[0210] 2. Cell uptake experiment of HeLa cells

[0211] The DNA molecules used in this experiment were DNA with Alexa flour 488 fluorescent modification at the 5' end (a custom product of Sangon Biotech (Shanghai) Co., Ltd., order number: 111989851, specific sequence shown in SEQ ID No. 8). The proteins used were SUMO-PRM1 and GE11-SUMO-PRM1. The specific sequence of SUMO-PRM1 is shown in SEQ ID No. 1, and the protein sequence of GE11-SUMO-PRM1 is shown in SEQ ID No. 5. The 59nt ssDNA sequence used is shown in SEQ ID No. 8. The mass ratio of GE11-SUMO-PRM1 to 59nt ssDNA was GE11-SUMO-PRM1:nucleic acid = 5:1, and the mass ratio of SUMO-PRM1 to 59nt ssDNA was SUMO-PRM1:nucleic acid = 4:1. Assembly was performed according to assembly method 2. The experimental groups are as follows:

[0212] (A) After adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 to HeLa cells and reacting for 2 hours, the cells were stained with Hoechst dye and observed.

[0213] (B) Two hours after adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to HeLa cells, the cells were stained with Hoechst dye and observed.

[0214] (C) After adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 to HeLa cells and reacting for 4 hours, the cells were observed after staining with Hoechst dye.

[0215] (D) After adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to HeLa cells and reacting for 4 hours, the cells were observed after staining with Hoechst dye.

[0216] (E) After adding nucleic acid-protein nanoparticles formed by SUMO-PRM1 to HeLa cells and reacting for 6 hours, the cells were observed after staining with Hoechst dye.

[0217] (F) After adding nucleic acid-protein nanoparticles formed by GE11-SUMO-PRM1 to HeLa cells and reacting for 6 hours, the cells were observed after staining with Hoechst dye.

[0218] (G) Add an equal amount of DNA to HeLa cells and incubate for 6 hours. Observe after staining with Hoechst dye.

[0219] Observations were conducted under the conditions of Merge, bright field, 488 nm wavelength excitation, and 346 nm wavelength excitation light. The results are as follows: Figure 7 As shown, compared to (G) where an equal amount of DNA was directly added, the results of (A), (B), (C), (D), and (E) indicate that nucleic acids were taken up by cells two hours after the addition of the particles, and the amount of nucleic acids taken up by cells increased over time. Therefore, it can be concluded that the particles can transport nucleic acids into HeLa cells. Furthermore, compared to SUMO-PRM1, the amount of particles formed by GE11-SUMO-PRM1 and nucleic acids taken up by cells was increased at the same time point, indicating that the N-terminus fusion of GE11 with PRM1 promotes the absorption of particles by HeLa cells, and the GE11 peptide can achieve targeted absorption of HeLa cells in vivo.

[0220] In conclusion, by adding a targeting peptide sequence to the N-terminus of a protein during recombinant expression, the uptake of particles by target cells can be enhanced, thereby enabling the targeted recognition of particles by target cells.

[0221] 3. HEK 293T cell uptake assay (the nucleic acids and proteins used were fluorescently labeled)

[0222] The DNA molecule used in this experiment was a 59 ntssDNA with Alexa flour 488 fluorescent modification at the 5' end (a custom product of Sangon Biotech (Shanghai) Co., Ltd., order number: 111989851, specific sequence see SEQ ID No. 8). The SUMO-PRM1 used was Alexa Fluorinated. TM Modified with 546 C5 (purchased from ThermoFisher, catalog number: A10258; see product manual for detailed usage instructions). Protein sequence is shown in SEQ ID No. 1.

[0223] The protein SUMO-PRM1 and 59 nt ssDNA were assembled at a mass ratio of 4:1 according to assembly method 2. The experimental groups are as follows:

[0224] (A) Observation was performed 8 hours after adding nucleic acid-protein nanoparticles to HEK 293T cells.

[0225] (B) Observation was performed 28 hours after adding nucleic acid-protein nanoparticles to HEK 293T cells.

[0226] Observations were conducted under Merge, bright field, 488 nm, and 564 nm excitation light conditions. Results are as follows: Figure 8 As shown, 8 hours after the addition of nanoparticles, cells were able to complete the uptake of the particles, and the uptake of particles by cells increased over time; at the same time, the observation results of excitation light at wavelengths of 488nm and 564nm indicate that the particles can not only transport nucleic acids into cells, but also transport proteins into cells.

[0227] In conclusion, these protein-nucleic acid nanoparticles can deliver proteins and nucleic acids into different cells.

[0228] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Protein-nucleic acid nanoparticles, wherein the protein-nucleic acid nanoparticles are nanoparticles made of nucleic acid molecules and protein molecules; wherein the protein molecules contain peptide segments, the length of the peptide segments is greater than or equal to 50 amino acid residues, the peptide segments contain greater than or equal to 12 positively charged amino acid residues, the positively charged amino acid residues being arginine residues; the peptide segments containing greater than or equal to 12 positively charged amino acid residues are protamine or a mutant of protamine; The amino acid sequence of the protein molecule is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5; The diameter of the protein and nucleic acid nanoparticles is 30 nm; The nucleic acid molecule is selected from at least one of the four types of nucleic acid molecules: single-stranded deoxyribonucleotide, double-stranded deoxyribonucleotide, single-stranded ribonucleotide, and double-stranded ribonucleotide; the size of the nucleic acid molecule is 21 bp-4733 bp; Based on protamine in the protein molecule, the mass ratio of the nucleic acid molecule to the protein molecule is 1:0.3-3.3; The nucleic acid molecules and the protein molecules are reacted in a high ionic strength buffer solution, and the resulting reaction product is reacted in a low ionic strength buffer solution to obtain the protein-nucleic acid nanoparticles. The high ionic strength buffer solution is composed of water, NaCl, and Tris-HCl, and the NaCl content in the high ionic strength buffer solution is 500-2000 mM. The low ionic strength buffer solution is composed of water, NaCl, and Tris-HCl, and the NaCl content in the low ionic strength buffer solution is 0-100 mM.

2. A method for preparing the protein-nucleic acid nanoparticles according to claim 1, comprising reacting the nucleic acid molecule and the protein molecule according to claim 1 in a high ionic strength buffer, reacting the resulting reaction product in a low ionic strength buffer, and obtaining the protein-nucleic acid nanoparticles; wherein the high ionic strength buffer is composed of water, NaCl, and Tris-HCl, and the NaCl content in the high ionic strength buffer is 500-2000 mM, and the low ionic strength buffer is composed of water, NaCl, and Tris-HCl, and the NaCl content in the low ionic strength buffer is 0-100 mM.

3. The method according to claim 2, characterized in that: Based on protamine in the protein molecule, the mass ratio of the nucleic acid molecule to the protein molecule is 1:0.3-3.

3.

4. The method according to claim 2, characterized in that: The reaction in the low ionic strength buffer solution includes reacting the reaction product first in the high ionic strength buffer solution with a NaCl content of 500-2000 mM, and then reacting it in the low ionic strength buffer solution with a NaCl content of 0-100 mM.

5. The application of the protein and nucleic acid nanoparticles according to claim 1 in the preparation of products that target and recognize tumor cells, wherein, The amino acid sequence of the protein molecule is SEQ ID NO.

5.

6. The use of the protein and nucleic acid nanoparticles of claim 1 in the preparation of products that deliver nucleic acids into cells.

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